Large molecules and detectable labels such as fluorophores, solid phase matrices or beads, antibodies, and hybridization probes are frequently attached to other molecules indirectly via linkers in order to avoid steric clashes which could decrease the affinity between those molecules and their targets, thus reducing their biological activity. A “linker” is a molecule that serves to join other atoms, molecules, or functional groups together via covalent or non-covalent interactions.
Ideally, a linker may separate an attached detectable label from a target molecule, without detracting from the useful properties of the detectable label, such as its signal intensity, binding affinity, or solubility. In practice, however, many linker molecules used in the art have a relatively short length, making it difficult to avoid steric clashes between the substances that they serve to juxtapose. For example, linkers such as 6-amino-hexanoic acid, succimidyl 4-(N-malemidomethyl)cylohexane-1-carboxylate (SMCC), and N-γ-malemidobutyryloxy succinimide ester (GMBS); are between 7 and 9 atoms in length, which would correspond to about 1 nm if they are fully extended in solution. That length is only a small fraction of the size of the average biological macromolecule such as a protein or oligonucleotide, and may be insufficient to relieve potential steric clashes between atoms, groups, or molecules to which those linkers are conjugated. Other examples of relatively short linkers used in the art include homobifunctional linkers such as glutaricdialdehyde, divinylsulfone, hexane diisocyanate, dimethylapimidate, 1,5-difluoro-2,4-dinitrobenzene, heterobifunctional linkers such as N-gamma-maleimidobytyroloxy succinimide ester, and zero length linkers such as 1-ethyl-3-(3-dimethylaminopropyl)cabodiimide.
Longer linker molecules based upon polyethylene glycol (PEG) are also available in the art. (See, for example, Discrete PEG (dPEG)™ modification reagents available from Quanta Biodesign, Ltd., Powell, Ohio, or at www.quantabiodesign.com; PEG-based reagents available from EMD Biosciences, Inc., San Diego, Calif., described in Novabiochem April, 2004, “Product focus: PEG reagents—bifunctional amino-PEG-acid spacers” brochure, available at www.novabiochem.com; and see Baumeister et al., Biopolymers, 71: 339 (2003); Kumar & Aldrich, Org. Lett., 5: 613 (2003). (See also, “Chemistry of Protein Conjugation and Cross-Linking” Shan S. Wong CRC Press, Boca Raton, Fla., USA, 1993; “BioConjugate Techniques” Greg T. Hermanson Academic Press, San Diego, Calif., USA, 1996; “Catalog of Polyethylene Glycol and Derivatives for Advanced PEGylation, 2004” Nektar Therapeutics Inc, Huntsville, Ala., USA.) While PEG-based linkers have a longer length, for example from 20 to over 70 atoms, they may not always remain extended in solution, and instead may aggregate or form unwanted tertiary structures. Such unwanted homo- or hetero-aggregation can negatively affect the activity of the conjugated atoms, groups, or molecules.
One object of the present invention was to find a linker structure that could adopt a variety of lengths, including very long lengths, that may have a well defined molecular and structural formula even at very long lengths, that would have less tendency to form unwanted tertiary structures or aggregates, that could readily and efficiently polymerize if desired, and that would exist in extended form in solution. The linkers of the present invention may allow for different conjugated atoms, groups, or molecules to be separated by short distances or by, for instance, up to several nanometers in solution. The instant linkers also may be designed such that they possess well defined molecular and structural formulas, providing greater uniformity to molecular entities that comprise them. In some of the instant embodiments, even very long linkers according to the present invention surprisingly remain in an extended and flexible structure in aqueous solutions and have little tendency to fold inward upon themselves, aggregate with each other, or form other tertiary structures that interfere with the functions of the conjugated atoms, groups, or molecules that they carry. Some of the instant linkers form chains with little steric bulk along their length, and thus may easily access narrow molecular spaces to interact with solid phase surfaces or membranes at a high density. The present invention also comprises methods of synthesizing the instant linkers and attaching them covalently to other molecules and functional groups at high yield.
The linkers of the present invention may be covalently or non-covalently attached to a variety of substances including color labels, components of solid surfaces, nucleic acids and nucleic acid analogs, proteins, and protein substrates. The ability of some of the instant linkers to remain extended and flexible in solution can minimize steric interference between the linker and the conjugated atom, group, or molecule, so that the conjugate's signal or its availability for recognition by target molecules is minimally perturbed by the linker. Thus, the present invention comprises a method of enhancing the signal intensity, activity, or binding affinity of a molecule such as a detectable label, including a probe, comprising conjugating the molecule to one or more of the instant linkers.
For example, when protein-nucleic acid (PNA) probes are conjugated to the linkers, the PNAs are less susceptible to aggregation and remain more available for binding to their intended target sequences. Further, because the structure of the instant linkers in solution may be both long and flexible, the instant linkers may be used to cross-link large molecules together in a way that allows the molecules to adopt their preferred orientations, and thus, minimally perturbs the structure and affinity of the molecular complex.
As another example, steric bulk or tertiary structure near a fluorescent label can cause quenching of the fluorescence due to absorbance of the signal by the surrounding molecular bonds. Interactions between two or more nearby fluorophores may also cause their signals to quench. This may be a problem in designing detectable labels comprising multiple conjugated fluorophores. For example, in protein detection, typically only about 3-4 fluorophores may be added before a plateau is reached in the intensity of the signals. The addition of further fluorophores may even diminish the overall signal. Such quenching may be reduced using embodiments of the present invention. Accordingly, this invention includes a method of enhancing the signal of one or more fluorophores, comprising conjugating the one or more fluorophores to at least one of the instant linkers. Because quenching due to the linker attachment of the present invention may be minimal, and because individual fluorophores may be conjugated so that they do not significantly interfere with each other, the present linkers also allow for the design of multiple-fluorophore linker derivatives having predictable emission colors that are a combination of the emissions from individual fluorophores of different emission spectra.
Another surprising feature of some of the present linkers is their high water solubility despite a relatively limited number of polar or charged groups. The instant linkers may enhance the solubility of a variety of different conjugated atoms, groups, or molecules. Thus, this invention also comprises a method of enhancing the solubility of a conjugated atom, group, or molecule, comprising directly or indirectly conjugating one or more atoms, groups, or molecules to one or more of the instant linkers.
Additional objects and advantages of the invention will be set forth in the description which follows, will be obvious from the description, or may be learned by practice of the invention. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.